Section Section O – RNA processing aO – RNA processing and RNPsnd RNPs

O1 rRNA processing and ribosomesO1 rRNA processing and ribosomes Types of RNA processing, rRNA processing in prokaryote,

rRNA processing in eukaryotes, RNPs and their study, Prokaryotic ribosomes, Eukaryotic ribosomes

O2 tRNA processing, RNase P and ribozyO2 tRNA processing, RNase P and ribozymesmes

tRNA processing in prokaryotes, tRNA processing in eukaryotes, RNase P, Ribozymes

O3 mRNA processing, hnRNPs and snRNPO3 mRNA processing, hnRNPs and snRNPss

Processing of mRNA, hnRNP, snRNP particles, 5’ Capping, 3’ Cleavage and polyadenylation, Splicing, Pre-mRNA methylation

O4 Alternative mRNA processingO4 Alternative mRNA processing Alternative processing, Alternative poly(A) site,

Alternative splicing, RNA editing

ContentsContents

O1 rRNA processing and ribosomes — O1 rRNA processing and ribosomes — Types of RNA processingTypes of RNA processing

• Very few RNA molecules are transcribed directly into the final mature RNA. Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product. RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

primary transcriptprimary transcript

mature RNAmature RNA..

Nucleus or Nucleolus

Cytoplasm

RN

A

pro

cessin

g

Romoval of nucleotides

addition of nucleotides to the 5’- or 3’- ends

modification of certain nucleotides

• (1) Removal of nucleotides by both endonucleases and exonucleases

• (2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products.

• (3) Modification of certain nucleotides on either the base or the sugar moiety.

O1 rRNA processing and ribosomes — O1 rRNA processing and ribosomes — rRNA processing in prokaryoterRNA processing in prokaryote1. There are 7 different operons for rRNA that are dis

persed throughout the genome.2. Each operon contains one copy of each of the 5S,t

he 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.

3. The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived.

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA

Pre-tRNA

Promoters TerminatorsrRNA operonrRNA operon

Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences

RNA folding

Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome

RNP complex formation

Step 3: After the binding of proteins, nucleotide modifications take place.

Example: methylation of adenine by methylating agent S-Adenosylmethonine (SAM)

Step 4: RNA cleavage

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA Pre-tRNA

Promoters Terminators

30S pre-rRNA: Transcription

Cleavage at

16S rRNA tRNA 23S rRNA 5S rRNA tRNA

RNase III III P F III III P F P ERNase III III P F III III P F P E

RNase M16 M16 M23 M23 M5RNase M16 M16 M23 M23 M5

rRNA operonrRNA operon

O1 rRNA processing and ribosomes — O1 rRNA processing and ribosomes — rRNA processing in eukaryotesrRNA processing in eukaryotes

• rRNA in eukaryotes is also generated from a single, long precursor molecule by specific modification and cleavage steps

• The processes are not so well understood

1. The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol I

2. Precursor sizes are different among organisms (yeast: 7000 nt; mammalian 13500 nt), and pre-mRNA processing is also slightly different among organism.

3. The precursor contains • one copy of the 18S coding region and • one copy each of the 5.8S and 28S coding

regions, which together are the equivalent of the 23S rRNA in prokaryote

4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.

5. The eukaryotic 5S rRNA

• is transcribed by RNA Pol III from unlinked genes to give a 121nt transcript

• the transcript undergoes little or no processing

18S rRNA 5.8S rRNA 28S rRNA

45S

41S

20S and 32S

Mature rRNAs

47S18S 5.8S 28S

ETS1 ITS1 ITS2 ETS2

Indicates RNase cleavage

Mammalian pre-rRNA processingMammalian pre-rRNA processing

• The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced.

• Mature rRNAs complex with protein to form RNPs (nucleolus)

• Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)

• Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced out to generate mature rRNAs.

• Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme

O1 rRNA processing and ribosomes — O1 rRNA processing and ribosomes — RNPs and their studyRNPs and their study

• Cells contain a variety of RNA-protein complexes( RNPs).

• These can be studied using techniques that help to clarify their structure and function.

• These include dissociation, re-assembly, electron microscopy, use of antibodies, RNase protection, RNA binding, cross-linking and neutron and X-ray diffraction.

• The structure and function of some RNPs are quite well characterized.

O1 rRNA processing and ribosomes — O1 rRNA processing and ribosomes — Prokaryotic ribosomesProkaryotic ribosomes• Protein biosynthetic

machinery• Made of 2 subunits

(bacterial 30S and 50S, & Eukaryotes 40S and 60S)

Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes

In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.

Features of the E.coli ribosome

Cleft

PlatformCentral protuberance

Small

Stalk

Ribosome Structure (1)

mRNA is associated with the 30S subunit

Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits.

The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.

Ribosome Structure (2)

O1 rRNA processing and ribosomes — O1 rRNA processing and ribosomes — Eukaryotic ribosomes Eukaryotic ribosomes

• larger and more complex than prokaryotic ribosomes, but with similar structural and functional properties

O2 tRNA processing, RNase P and ribozymes — O2 tRNA processing, RNase P and ribozymes — tRNA processing in prokaryotestRNA processing in prokaryotes• Mature tRNAs are generated by processing longer pre-t

RNA transcripts, which involves • specific exo- and endonucleolytic cleavage by RNases D,

E, F and P (general) followed by • base modifications which are unique to each particular t

RNA type.

O2 tRNA processing, RNase P and ribozymes — O2 tRNA processing, RNase P and ribozymes — tRNA processing in eukaryotestRNA processing in eukaryotes

• The pre-tRNA is synthesized with a 16 nt 5’-leader a The pre-tRNA is synthesized with a 16 nt 5’-leader a 14 nt intron and two extra 3’-nucleotides.14 nt intron and two extra 3’-nucleotides.

1. Primary transcripts forms secondary structures recognized by endonucleases

2. 5’ leader and 3’ extra nucleotide removal

3. tRNA nucleptidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end

4. Intron removal

O2 tRNA processing, RNase P and ribozymes — O2 tRNA processing, RNase P and ribozymes — RNase PRNase P• Ribonuclease P (RNase P) is an enzyme involve

d in tRNA processing that removes the 5' leader sequences from tRNA precursors

• RNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)

• In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa.

• RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

O2 tRNA processing, RNase P and ribozymes — O2 tRNA processing, RNase P and ribozymes — RibozymesRibozymes• Ribozymes are catalytic RNA molecules that can catalyz

e particular biochemical reactions.• RNase P RNA is a ribozyme.• RNase P RNA from bacteria is more catalytically active i

n vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures.

• Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences

• Group I introns, such as Tetrahymena intron• Group II introns.

• Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA

• HDV ribozyme• Hairpin ribozyme• Hammer head ribozyme• Ribozymes can be used as therapeutic agents in • correcting mutant mRNA in human cells• inhibiting unwanted gene expression• Kill cancer cells• Prevent virus replication

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — Processing of mRNAProcessing of mRNA• Processing of mRNA: prokaryotes • There is essentially no processing of prokaryotic mRNA,

it can start to be translated before it has finished being transcribed.

• Prokaryotic mRNA is degraded rapidly from the 5’ end• Processing of mRNA in eukaryotes• In eukaryotes, mRNA is synthesized by RNA Pol II as lo

nger precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA).

• Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs

Eukaryotic mRNA processing: overview

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — hnRNPhnRNP• The hnRNA synthesized by RNA Pol II is

mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP)

• The hnRNP proteins are though to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — snRNP particlessnRNP particles

1. snRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs.

2. The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6.

3. A large number of snRNP define methylation sites in pre-rRNA.

4. snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap.

5. Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.

6. Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — 5’ Capping5’ Capping

• Very soon after RNA Pol II starts making a transcript, and before the RNA chain is more then 20 -30 nt long, the 5’-end is chemically modified.

• 7-methylguanosine is covalently to the 5´ end of pre-mRNA.

• Linked 5´ 5´

• Occurs shortly after initiation

7-methylguanosine (m7G)

Function of 5´cap• Protection from degradation

• Increased translational efficiency

• Transport to cytoplasm

• Splicing of first exon

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — 3’ Cleavage and polyadenylation3’ Cleavage and polyadenylation• In most pre-mRNAs, the mature 3’-end of the mo

lecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.

• RNA polymerase II does not usually terminate at distinct site

• Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)

• ~250 AMPs are then added to the 3´ end• Almost all mRNAs have poly(A) tail

Function of poly(A) tail

• Increased mRNA stability

• Increased translational efficiency

• Splicing of last intron

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — SplicingSplicing• the process of cutting the pre-mRNA to remove the intro

ns and joining together of the exons is called splicing.• it takes place in the nucleus before the mature mRNA ca

n be exported to the cytoplasm.• Introns: non-coding sequences• Exons: coding sequences• RNA splicing: removal of introns and joining of exons• Splicing mechanism must be precise to maintain open re

ading frame• Catalyzed by spliceosome (RNA + protein)

Biochemical steps of pre-mRNA splicing

Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site

Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.

C U R A Y

Lariat

Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.

Spliceosome• Catalyzes pre-mRNA splicing in nucleus• Composed of five snRNPs (U1, U2, U4, U5

and U6), other splicing factors, and the pre-mRNA being assembled

• U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.

• U2 and U6 snRNA are able to catalyze the splicing reaction.

Splicing cycle

O3 mRNA processing, hnRNPs and snRNPs — O3 mRNA processing, hnRNPs and snRNPs — Pre-mRNA methylationPre-mRNA methylation

• The final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases.

• The methylations seem to be largely conserved in the mature mRNA.

O4 Alternative mRNA processing — O4 Alternative mRNA processing —

Alternative processingAlternative processing

• Alternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.

• Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.

O4 Alternative mRNA processing — O4 Alternative mRNA processing — Alternative poly(A) siteAlternative poly(A) site

• Some pre-mRNAs contain more than one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.

• In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.

O4 Alternative mRNA processing — O4 Alternative mRNA processing —

Alternative splicingAlternative splicing

• The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways:

• By using different promoters

• By using different poly(A) sites

• By retaining certain introns

• By retaining or removing certain exons

Alternative splicing

(A) A cassette exon can be either included in the mRNA or excluded.

(B) Mutually exclusive exons occur when two or more adjacent cassette exons are spliced such that only one exon in the group is included at a time.

(C, D) Alternative 5’ and 3’ splice sites allow the lengthening or shortening of a particular exon.

(E, F) Alternative promoters and alternative poly(A) sites switch the 59- or 39-most exons of a transcript.

(G) A retained intron can be excised from the pre-mRNA or can be retained in the translated mRNA.

(H) A single pre-mRNA can exhibit multiple sites of alternative splicing using different patterns of inclusion.

Alternative splicing

O4 Alternative mRNA processing — O4 Alternative mRNA processing — RNA RNA editingediting• An unusual form of RNA processing in whi

ch the sequence of the primary transcript is altered is called RNA editing.

• Changing RNA sequence (after transcription)

• RNA editing is known to occur in two different situations, with different causes.

• In mammalian cells there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded. (Base modification:A or C deamination)

• In trypanosome mitochondria, more widespread changes occur in transcripts of several genes, when bases are systematically added or deleted. (Base U insertion and deletion)

Multiple choice Multiple choice questionsquestions

1. Which of the following terms correctly describe parts of the E. coli large (50S) subunit?

A stalk central protuberance valley and cleft. B upper third lower third valley and stalk. C cleft valley stalk and small protuberance. D stalk polypeptide exit site valley and central protuberance.2. Which ribonucleases are involved in producing mature tRNA in

E. coli?A RNases A, D, E and F. B RNases D, E, F and H. C RNases D, E, F and P. D RNases A, D, H and P.

3. Most eukaryotic pre-mRNAs are matured by which of the following modifications to their ends?

A capping at the 3’-end cleavage and polyadenylation at the 5'-end. B addition of a GMP to the 5'-end ， cleavage and polyadenylation to create the 3'-end. C addition of a guanine residue to the 5'-end cleavage and polyadenylation to create the

3'-end. D addition of a GMP to the 5'-end ， polyadenylation ， then cleavage to create the 3'-e

nd.

4. Which one of the following statements correctly describes the splicing process undergone by most eukaryotic pre-mRNAs?

A in a two-step reaction, the spliceosome removes the exon as a lariat and joins the two introns together.

B splicing requires conserved sequences which are the 5ιsplice site ， the 3' -splice site the branch-point and the polypurine tract.

C the U1 snRNP initially binds to the 5'-splice site ， U2 to the branchpoint sequence and then the tri-snRNP, U4, US and U6 can bind.

D in the first step of splicing the G at the 3'-end of the intron is joined to the 2’-hydroxyl group of the A residue of the branchpoint sequence to create a lariat.

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